This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To provide expert support to AIDS research conducted at the Primate Center. 1. PROGRESS AND CONCERNS: The Virology Services Unit (VS) performed over 1,700 SIV virus load determinations in FY2009. This is fewer than in FY2008, as our core users pared back on the scope of their studies. We anticipate increased demand in FY2010 as established users are funded to perform more and larger experiments. In anticipation of this increased demand, we tested a new workflow using the high-throughput Roche LightCycler 480 quantitative PCR platform in February 2010. Transition to this workflow will facilitate an expansion of our services and will simplify batch processing of samples as we now charge all users, both intra- and extramural, for virus load determinations. Our chargeback system for extramural investigators had 2 users and generated revenues of $18,255. VS also produced over 4,000 vials of high-titer SIVmac239 stock and 3 custom SIV mutants for WNPRC investigators. In response to investigators interested in evaluating the fitness of multiple mutant strains of SIV, we deployed a "barcode virus" reporter system. We used site-directed mutagenesis to insert a genetic "barcode" of silent mutations into the region of SIVmac239 gag targeted by our standard QRT-PCR assay. These mutations do not affect virus fitness, but they abrogate detection using the primers and probes of our standard assay. This "barcode" virus thus provides a standard against which any viral variant can be directly compared in competing coculture assays. The Tetramer Core of Immunology Services Unit (IS) produced approximately 99,000 tetramer tests in 2009. 25,508 tests were shipped to ten different investigators at nine different institutions, bringing in $76,524 in chargeback income. We also distributed 7,155 tests to laboratories on campus, including the David H. O'Connor laboratory in Pathology, the Thomas Friedrich laboratory at the Vet School and the Watkins Laboratory in Pathology. All three of these labs are also part of the WNPRC. Our FPLC instruments were also utilized by non-primate center investigators, assisting them in preliminary studies, or when their FPLC was not working. We are currently pursuing MHC class II tetramers, and expect to produce our first tetramers in early 2010. We have expanded the alleles that are available for tetramer production in 2009, to include several cynomolgus alleles. We will continue expanding our allele availability in 2010, as more and more cyno and rhesus alleles are found to be important in SIV studies. In addition, we are developing Elisas for class II tetramers, as well as a diagnostic test for peptide binding. Our group makes all of the p27 antibody used by our lab, and also shipped 200 tests of this reagent to Stephen Kent's lab in Australia. For extramural investigators more than 1200 blood samples were processed and shipped out, and 1680 IFN-[unreadable] Elispot tests and 327 intracellular cytokine staining tests were performed. IS also provides expert support to Primate Center, UW-Madison and extramural investigators wishing to use our flow cytometry facilities. More than 2,500 hours were used for flow data acquisition during this year. These activities together resulted in an additional ~$55,000 in chargebacks. In 2009 we purchased a new custom-made BD-LSR II machine to expand our services. We have established or supported the development of several multicolor staining protocols. These included staining panels to test different functional capabilities of antigen specific T cell populations, and panels that identify innate immune responses after yellow fever vaccination. To promote better data analysis in flow cytometry we have introduced the use of Pestle and Spice data analysis softwares. To expand our services with BL-3 level sorting capabilities Dr Rakasz will be trained to operate a FACSAria high speed cell sorter in February. She will have access to a BL-3 level sorting facility on campus and will perform cell sort for investigators who has this request. 2. ALLOCATION OF RESOURCE ACCESS: The central mission of IVS is to provide expert support to AIDS and infectious disease related research conducted at the Primate Center by WNPRC or outside investigators. In fiscal 2009 IS served 9 on-campus and 15 off-campus laboratories, which were funded by both federal and non-federal grants. VS supported 4 on-campus and 2 off-campus users in FY2009. 3. DISSEMINATION: Drs. Friedrich, Wilson and Rakasz, the PIs of VS and IS units, consult closely with users of the service, helping to design experiments and interpret results. We request that projects utilizing Virology and Immunology Services acknowledge the service in manuscripts and presentations. 4. TRAINING: Dr. Friedrich consults regularly with recognized leaders in SIV virology and molecular biology to develop and refine our techniques. For example, custom SIV mutagenesis methods were developed in collaboration with Dr. Ronald Desrosiers at the New England Primate Center. Quantitative RT-PCR techniques were developed in consultation with Dr. Jeffrey Lifson at the National Cancer Institute. IS staff have been trained in flow cytometry techniques at Beckton Dickinson. Dr Rakasz and Ms Kim Weisgrau, members of the flow cytometry facility regularly train new investigators to acquire their data independently on the flow machines at their disposal. PUBLICATIONS: Bonaldo MC, Martins MA, Rudersdorf R, Mudd PA, Sacha JB, Piaskowski SM, Costa Neves PC, Veloso de Santana MG, Vojnov L, Capuano S 3rd, Rakasz EG, Wilson NA, Fulkerson J, Sadoff JC, Watkins DI, Galler R. Recombinant Yellow Fever Vaccine Virus 17D Expressing SIVmac239 Gag Induces SIV-Specific CD8+ T Cell Responses in Rhesus Macaques. J Virol. 2010 Jan 20. [Epub ahead of print] PMID: 20089645 Maness NJ, Wilson NA, Reed JS, Piaskowski SM, Sacha JB, Walsh AD, Thoryk E, Heidecker GJ, Citron MP, Liang X, Bett AJ, Casimiro DR, Watkins DI. Robust, vaccine-induced CD8(+) T lymphocyte response against an out-of-frame epitope. J Immunol. 2010 Jan 1;184(1):67-72. PMID: 19949108 Hessell AJ, Rakasz EG, Tehrani DM, Huber M, Weisgrau KL, Landucci G, Forthal DN, Koff WC, Poignard P, Watkins DI, Burton DR. Broadly neutralizing monoclonal antibodies 2F5 and 4E10 directed against the human immunodeficiency virus type 1 gp41 membrane-proximal external region protect against mucosal challenge by simian-human immunodeficiency virus SHIVBa-L. J Virol. 2010 Feb;84(3):1302-13..PMID: 19906907 Vojnov L, Reed JS, Weisgrau KL, Rakasz EG, Loffredo JT, Piaskowski SM, Sacha JB, Kolar HL, Wilson NA, Johnson RP, Watkins DI. Effective simian immunodeficiency virus-specifici CD8+ T cells lack an easily detectable, shared characteristic. J Virol. 2010 Jan;84(2):753-64. PMID: 19889785 Valentine LE, Loffredo JT, Bean AT, Le[unreadable]n EJ, MacNair CE, Beal DR, Piaskowski SM, Klimentidis YC, Lank SM, Wiseman RW, Weinfurter JT, May GE, Rakasz EG, Wilson NA, Friedrich TC, O'Connor DH, Allison DB, Watkins DI. Infection with "escaped" virus variants impairs control of simian immunodeficiency virus SIVmac239 replication in Mamu-B*08-positive macaques. J Virol. 2009 Nov;83(22):11514-27. PMID: 19726517 Maness NJ, Sacha JB, Piaskowski SM, Weisgrau KL, Rakasz EG, May GE, Buechler MB, Walsh AD, Wilson NA, Watkins DI. Novel translation products from simian immunodeficiency virus SIVmac239 Env-encoding mRNA contain both Rev and cryptic T-cell epitopes. J Virol. 2009 Oct;83(19):10280-5. PMID: 19605480 Loffredo JT, Sidney J, Bean AT, Beal DR, Bardet W, Wahl A, Hawkins OE, Piaskowski S, Wilson NA, Hildebrand WH, Watkins DI, Sette A. Two MHC class I molecules associated with elite control of immunodeficiency virus replication, Mamu-B*08 and HLA-B*2705, bind peptides with sequence similarity. J Immunol. 2009 Jun 15;182(12):7763-75.PMID: 19494300. Sacha JB, Giraldo-Vela JP, Buechler MB, Martins MA, Maness NJ, Chung C, Wallace LT, Le[unreadable]n EJ, Friedrich TC, Wilson NA, Hiraoka A, Watkins DI. Gag- and Nef-specific CD4+ T cells recognize and inhibit SIV replication in infected macrophages early after infection. Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9791-6. PMID: 19478057 Hessell AJ, Rakasz EG, Poignard P, Hangartner L, Landucci G, Forthal DN, Koff WC, Watkins DI, Burton DR. Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers. PLoS Pathog. 2009 May;5(5):e1000433. PMID: 19436712 Wilson NA, Keele BF, Reed JS, Piaskowski SM, MacNair CE, Bett AJ, Liang X, Wang F, Thoryk E, Heidecker GJ, Citron MP, Huang L, Lin J, Vitelli S, Ahn CD, Kaizu M, Maness NJ, Reynolds MR, Friedrich TC, Loffredo JT, Rakasz EG, Erickson S, Allison DB, Piatak M Jr, Lifson JD, Shiver JW, Casimiro DR, Shaw GM, Hahn BH, Watkins DI. Vaccine-induced cellular responses control simian immunodeficiency virus replication after heterologous challenge. J Virol. 2009 Jul;83(13):6508-21. PMID: 19403685 Greene JM, Lhost JJ, Burwitz BJ, Budde ML, Macnair CE, Weiker MK, Gostick E, Friedrich TC, Broman KW, Price DA, O'Connor S, O'Connor DH. Extra-lymphoid tissue-resident CD8+ T cells from SIVmac239Deltanef-vaccinated macaques suppress SIVmac239 replication ex vivo. J Virol. 2010 Jan 20. Burwitz BJ, Pendley CJ, Greene JM, Detmer AM, Lhost JJ, Karl JA, Piaskowski SM, Rudersdorf RA, Wallace LT, Bimber BN, Loffredo JT, Cox DG, Bardet W, Hildebrand W, Wiseman RW, O'Connor SL, O'Connor DH. Mauritian cynomolgus macaques share two exceptionally common major histocompatibility complec class I alleles that restrict simian immunodeficiency virus specific CD8+ T cells. J Virol. 2009 Jun;83(12):6011-9. PMID: 19339351 Choi KD, Vodyanik MA, Slukvin II. Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell-derived lin-CD34+CD43+CD45+ progenitors. J Clin Invest. Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA. Human induced pluripotent stem cells free of vector and transgene sequences. Science. 2009 May 8;324(5928):797-801. PMID: 19325077 Rozner AE, Dambaeva SV, Drenzek JG, Durning M, Golos TG.Generation of macrophages from peripheral blood monocytes in the rhesus monkey. J Immunol Methods. 2009 Dec 31;351(1-2):36-40. PMID: 19818793 Bondarenko GI, Dambaeva SV, Grendell RL, Hughes AL, Durning M, Garthwaite MA, Golos TG. Characterization of cynomolgus and vervet monkey placental MHC class I expression: diversity of the nonhuman primate AG locus. Immunogenetics. 2009 Jun;61(6):431-42. PMID: 19468726 Dambaeva SV, Breburda EE, Durning M, Garthwaite MA, Golos TG. Characterization of decidual leukocyte populations in cynomolgus and vervet monkeys. J Reprod Immunol. 2009 Jun;80(1-2):57-69. PMID: 19398130 Drenzek JG, Vidiguriene J, Vidiguris G, Grendell RL, Dambaeva SV, Durning M, Golos TG. Suppression of Mamu-AG by RNA interference. Am J Reprod Immunol. 2009 Apr 22. PMID: 19392979 Li Q, Skinner PJ, Duan L, Haase AT. A technique to simultaneously visualize virus-specific CD8+ T cells and virus-infected cells in situ. J Vis Exp. 2009 Aug 13;(30). pii: 1561. doi: 10.3791/1561. Salisch NC, Kaufmann DE, Awad AS, Reeves RK, Tighe DP, Li Y, Piatak M Jr, Lifson JD, Evans DT, Pereyra F, Freeman GJ, Johnson RP. Inhibitory TCR coreceptor PD-1 is a sensitive indicator of low-level replication of SIV and HIV-1. J Immunol. 2010 Jan 1;184(1):476-87.